Compositions and methods for the repair of myelin

ABSTRACT

Described are oxysterols, pharmaceutical compositions including the oxysterols, and methods of using the oxysterols and compositions for treating diseases and/or disorders related to myelin injury, such as neonatal brain injury, traumatic brain injury, spinal cord injury, cerebral palsy, seizures, cognitive delay, multiple sclerosis, stroke, autism, leukodystrophy, schizophrenia and bipolar disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/022,261, filed Jul. 9, 2014, which is herein incorporated byreference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compounds, compositions, and methodsfor treating diseases and/or disorders related to myelin injury, such asneonatal brain injury, traumatic brain injury, spinal cord injury,cerebral palsy, seizures, cognitive delay, multiple sclerosis, stroke,autism, leukodystrophy, schizophrenia and bipolar disorder.

BACKGROUND

About 10% of infants in the United States are born premature and are atsignificant risk for brain injuries, which may lead to disorders such ascerebral palsy, seizures, and cognitive delay. Myelin injury is the mostcommon form of brain injury impacting neurodevelopment in prematureinfants. Currently there are no effective therapies for myelin injuries.

Human breast milk is associated with improved cognitive development.Recent studies have shown that a direct correlation between duration ofbreast-feeding and the integrity of myelination microstructure exists inhumans. However, the mechanisms of breast milk-associated improvementsin myelin development are unknown.

Accordingly, there exists a need for effective therapies for braininjuries resulting from damaged myelin. In addition, investigation intothe mechanisms of breast milk-associated improvements in myelindevelopment may reveal strategies for therapeutic intervention ininjured myelin related diseases and/or disorders in infants.

SUMMARY OF THE INVENTION

In one aspect, disclosed is a method of promoting oligodendrogenesis ina subject in need thereof, the method comprising administering atherapeutically effective amount of at least one oxysterol.

In another aspect, disclosed is a method of repairing injured myelin ina subject in need thereof, the method comprising administering atherapeutically effect amount of an oxysterol.

Also disclosed are pharmaceutical compositions comprising the compounds,and methods of using the pharmaceutical compositions for treatment ofdiseases and/or disorders related to myelin injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a series of confocal micrographs showing differentiated cellsstained for oligodendrocyte markers (OLIG2 and CNPase).

FIG. 1B is a magnification of a confocal micrograph of FIG. 1A showingoligodendrocyte morphology.

FIG. 1C is a graph illustrating the increase in oligodendrocyteformation after treatment with 20α-hydroxycholesterol.

FIG. 1D is a Western blot analysis showing an increase inoligodendrogenesis biomarkers after treatment with20α-hydroxycholesterol.

FIG. 2A is a picture of a tissue section of the corpus callosum of avehicle treated mouse stained for Olig2.

FIG. 2B is a picture of a tissue section of the corpus callosum of amouse treated with 20α-hydroxycholesterol stained for Olig2.

FIG. 2C is a graph illustrating the increase in oligodendrocyteformation after treatment with 20α-hydroxycholesterol.

FIG. 3A is a diagram of the experimental design used to test oxysteroltherapy in sepsis injured perinatal mice.

FIG. 3B is a panel of confocal images of Olig2+APC+ cells within thecorpus callosum of control sepsis, and sepsis+ oxysterol treated mice.

FIG. 3C is a graph illustrating the increase in oligodendrocyteformation after treatment of mice having sepsis injury with20α-hydroxycholesterol in comparison to vehicle treated mice with andwithout sepsis injury.

FIG. 4A-4C are plots of gait measurements after treatment of mice havingsepsis injury with 20α-hydroxycholesterol in comparison to vehicletreated mice with and without sepsis injury. FIG. 4B is a graphillustrating the Regularity Index resulting from the gait measurements.

FIG. 5A-5D are graphs depicting the results of Stand and Step cyclemeasurements and swing and swing speed meaurements after treatment ofmice having sepsis injury with 20α-hydroxycholesterol in comparison tovehicle treated mice with and without sepsis injury.

FIG. 6A is a graph showing results of mass spectrometry experiments toanalyze oxysterol standards.

FIG. 6B is a graph depicting the measurement of different oxysterols inhuman breast milk.

FIG. 7 is a graph depicting the measurement of different oxysterolconcentrations in human breast milk.

FIG. 8 is a western blot analysis. Stem cells were treated withoxysterols at doses indicated for 5 days then allowed to differentiatefor 18 days. Protein lysates were probed for oligodendrocyte-associatedproteins CNPase and myelin basic protein (MBP).

DETAILED DESCRIPTION

Disclosed herein are oxysterols useful for the treatment of disordersand diseases related to injury to myelin. The disclosed oxysterols areoxidized derivatives of cholesterol. The disclosed oxysterols can beused to repair injured myelin by promoting oligodendrogenesis fromneural stem cells and/or oligodendrocyte precursor cell populations.Injured myelin has been implicated in a number of different diseases anddisorders including, but not limited to, neonatal brain injury,traumatic brain injury, spinal cord injury, cerebral palsy, seizures,cognitive delay, multiple sclerosis, stroke, autism, leukodystrophy,schizophrenia and bipolar disorder.

Initially, a perinatal mouse model of myelin injury was developed, whichrecapitulated the pathological features and motor dysfunction observedin cerebral palsy. Employment of this model led to the discovery of aninjury to the cerebral neural stem cell population. Using lineagetracing experiments, it was discovered that these neural stem cellsstopped producing oligodendrocytes and began producing astrocytes inresponse to injury. Spurred by these observations, it was postulatedthat there may be biological targets within the stem cell populationthat could redirect these stem cells back into the oligodendrocytelineage and promote oligodendrocyte differentiation.

For example, the sonic hedgehog (SHH) signaling pathway has been shownto promote oligodendrogenesis in vitro and in vivo. In addition,oxysterols are natural ligands for the SHH pathway and recent studiesdemonstrated in fibroblast cell culture systems that the oxysterols,20α-hydroxycholesterol and 22α-hydroxycholesterol, can activate the SHHpathway via direct binding to smoothened (SMO). Once SMO is bound to theoxysterol, it is believed that the negative regulator Patchedl (PTCH1)cannot interact with SMO, resulting in SHH pathway activation andsubsequent oligodendrogenesis.

Accordingly, compounds that promote oligodendrogenesis, such as theoxysterols of the present disclosure can be useful in treating diseasesrelated to myelin pathology.

Furthermore, human breast milk may be an appropriate vehicle for theadministration of oxysterol therapy to infants in need of such therapy.Multiple naturally occurring oxysterols are identified in human breastmilk, increasing the viability of employing its use in therapies forneonatal brain injuries and related disorders and diseases.

In addition, most infants born prematurely are typically administeredhuman breast milk for nourishment. Because oxysterols can be used topromote oligodendrogenesis and healthy myelin, breast milk or infantformula supplemented with additional amounts of oxysterols may bebeneficial to promoting brain development in prematurely born infants,regardless of suspected or known brain injury.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The term “oxidized derivative” as used herein, means a compoundsubstituted with an oxygen containing group, such as, but not limitedto, at least one of a hydroxyl, oxo, alkoxy, epoxy or carboxy group.

The term “parenterally,” as used herein, refers to modes ofadministration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “an” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). The modifier “about” shouldalso be considered as disclosing the range defined by the absolutevalues of the two endpoints. For example, the expression “from about 2to about 4” also discloses the range “from 2 to 4.” The term “about” mayrefer to plus or minus 10% of the indicated number. For example, “about10%” may indicate a range of 9% to 11%, and “about 1” may mean from0.9-1.1. Other meanings of “about” may be apparent from the context,such as rounding off, so, for example “about 1” may also mean from 0.5to 1.4.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this disclosure, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

In some instances, the number of carbon atoms in a hydrocarbylsubstituent (e.g., alkyl or cycloalkyl) is indicated by the prefix“C_(x)-C_(y)-”, wherein x is the minimum and y is the maximum number ofcarbon atoms in the substituent. Thus, for example, “C₁-C₃-alkyl” refersto an alkyl substituent containing from 1 to 3 carbon atoms.

For compounds described herein, groups and substituents thereof may beselected in accordance with permitted valence of the atoms and thesubstituents, such that the selections and substitutions result in astable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. Compounds

A. Oxysterols

Oxysterols are oxidized derivatives of cholesterol. Oxysterols usefulfor the methods and compositions of the present disclosure may beoxidized derivatives of cholesterol wherein cholesterol is oxidized atany carbon of cholesterol. Oxysterols useful for the methods andcompositions of the present disclosure may be substituted with an oxygencontaining group, such as, but not limited to, at least one of ahydroxyl, oxo, alkoxy, epoxy or carboxy group.

Cholesterol

Oxysterols may be important in many biological processes, includingcholesterol homeostasis, atherosclerosis, sphingolipid metabolism,platelet aggregation, apoptosis, and protein prenylation, though theirroles are often poorly understood. Oxysterols are lipophilic and crossthe blood brain barrier. They are naturally present in small amounts inthe brain and they are known ligands for the Liver X Receptor (LXR) andSonic Hedgehog (SHH) signaling pathways. Oxysterols may be oxidized atsites on the tetracyclic ring structure or on the C20-27 aliphaticchain. Specific oxysterols include the following:

In certain embodiments, the oxysterol may be selected from the groupconsisting of:

20α-hydroxycholesterol;

22(R)-hydroxycholesterol;

22(S)-hydroxycholesterol;

24(R)-hydroxycholesterol;

24(S)-hydroxycholesterol;

25-hydroxycholesterol; and

27-hydroxycholesterol; or a pharmaceutically acceptable salt thereof

The compound may exist as a stereoisomer wherein asymmetric or chiralcenters are present. The stereoisomer is “R” or “S” depending on theconfiguration of substituents around the chiral carbon atom. The terms“R” and “S” used herein are configurations as defined in IUPAC 1974Recommendations for Section E, Fundamental Stereochemistry, in PureAppl. Chem., 1976, 45: 13-30. The disclosure contemplates variousstereoisomers and mixtures thereof and these are specifically includedwithin the scope of this invention. Stereoisomers include enantiomersand diastereomers, and mixtures of enantiomers or diastereomers.Individual stereoisomers of the compounds may be prepared syntheticallyfrom commercially available starting materials, which contain asymmetricor chiral centers or by preparation of racemic mixtures followed bymethods of resolution well-known to those of ordinary skill in the art.These methods of resolution are exemplified by (1) attachment of amixture of enantiomers to a chiral auxiliary, separation of theresulting mixture of diastereomers by recrystallization orchromatography and optional liberation of the optically pure productfrom the auxiliary as described in Furniss, Hannaford, Smith, andTatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5th edition(1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2)direct separation of the mixture of optical enantiomers on chiralchromatographic columns or (3) fractional recrystallization methods.

It should be understood that the compound may possess tautomeric forms,as well as geometric isomers, and that these also constitute an aspectof the invention.

The present disclosure also includes an isotopically-labeled compound,which is identical to the recited oxysterols, but for the fact that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes suitable for inclusion in the compounds ofthe invention are hydrogen, carbon, nitrogen, oxygen, phosphorus,sulfur, fluorine, and chlorine, such as, but not limited to ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.Substitution with heavier isotopes such as deuterium, i.e., ²H, canafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, may be preferred in some circumstances. Thecompound may incorporate positron-emitting isotopes for medical imagingand positron-emitting tomography (PET) studies for determining thedistribution of receptors. Suitable positron-emitting isotopes that canbe incorporated in the disclosed oxysterols are ¹¹C, ¹³N, ¹⁵O, and ¹⁸F.Isotopically-labeled oxysterols can generally be prepared byconventional techniques known to those skilled in the art or byprocesses analogous to those described in the accompanying Examplesusing appropriate isotopically-labeled reagent in place ofnon-isotopically-labeled reagent.

A. Oligodendrocytes and Oligodendrogenesis

Oxysterols of the present disclosure can promote the differentiation ofoligodendrocytes from neural stem cells.

Oligodendrocytes are a type of neuroglia. They function to providesupport and insulation to axons in the central nervous system bycreating the myelin sheath. Oligodendrocytes arise during developmentfrom oligodendrocyte precursor cells. Most oligodendrocytes developduring embryogenesis and early postnatal life from restrictedperiventricular germinal regions.

Oligodendrocytes are found in the central nervous system (CNS) andoriginate from the ventral ventricular zone of the embryonic spinalcord. They are the last cell type to be generated in the CNS.Myelination is only prevalent in a few brain regions at birth andcontinues into adulthood. The entire process is not complete until about25-30 years of age.

As part of the nervous system, oligodendrocytes are closely related tonerve cells and provide a supporting role for neurons. In addition, thenervous system of mammals depends on myelin sheaths, which reduce ionleakage and decrease the capacitance of the cell membrane. Myelin alsoincreases impulse speed, as saltatory propagation of action potentialsoccurs at the nodes of Ranvier in between Schwann cells (of the PNS) andoligodendrocytes (of the CNS). Myelinating oligodendrocytes are a partof the white matter and myelination is an important component ofintelligence.

1. Biomarkers of Oligodendrogenesis

Oligodendrogenesis may be determined by measuring the concentration ofcertain biomarkers in tissue. These biomarkers include OligodendrocyteTranscription Factor (OLIG2), 2′, 3′-Cyclic-Nucleotide3′-Phosphodiesterase (CNPase), and Myelin Basic Protein (MBP). Thepresence of, or an increase in the concentration of these biomarkers mayindicate oligodendrocyte formation.

a. Oligodendrocyte Transcription Factor

Oligodendrocyte transcription factor (OLIG2) is a basic helix-loop-helixtranscription factor encoded by the Olig2 gene. The protein is of 329amino acids in length, 32 kDa in size and contains 1 basichelix-loop-helix DNA-binding domain. The expression of OLIG2 is mostlyrestricted in central nervous system, and is well known for determiningoligodendrocyte differentiation.

OLIG2 is mostly expressed in restricted domains of the brain and spinalcord ventricular zone which give rise to oligodendrocytes and specifictypes of neurons. During embryogenesis, OLIG2 first directs motor neuronfate by establishing a ventral domain of motor neuron progenitors andpromoting neuronal differentiation. OLIG2 then switches to promoting theformation of oligodendrocyte precursors and oligodendrocytedifferentiation at later stages of development.

b. 2′, 3′-Cyclic-nucleotide 3′-phosphodiesterase

2′, 3′-Cyclic-nucleotide 3′-phosphodiesterase (CNPase) is amyelin-associated enzyme that makes up 4% of total CNS myelin protein,and is thought to undergo significant age-associated changes. It isnamed for its ability to catalyze the phosphodiester hydrolysis of 2′,3′-cyclic nucleotides to 2′-nucleotides, though a cohesive understandingof its specific physiologic functions are still ambiguous.

CNPase is expressed exclusively by oligodendrocytes in the CNS, and theappearance of CNPase seems to be one of the earliest events ofoligodendrocyte differentiation. CNPase may play a critical role in theevents leading up to myelination.

c. Myelin Basic Protein

Myelin basic protein (MBP) is important in the process of myelination ofnerves in the nervous system. The myelin sheath is a multi-layeredmembrane, unique to the nervous system, that functions as an insulatorto greatly increase the velocity of axonal impulse conduction. MBPmaintains the correct structure of myelin, interacting with the lipidsin the myelin membrane.

The disclosed oxysterols can promote the formation of oligodendrocytessuch that a treated subject has an increase of oligodendrocyteformation. The increase in oligodendrocyte formation may be measuredrelative to oligodendrocyte levels pretreatment in the subject. Theincrease of oligodendrocyte formation may be measured relative tooligodendrocyte levels in an untreated subject. The increase inoligodendrocyte formation may be measured relative to oligodendrocytelevels in an untreated control.

The disclosed oxysterols may promote an increase in oligodendrocyteformation of at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at least 110%, at least 120%, at least 130%, at least 140%,at least 150%, at least 160%, at least 170%, at least 180%, at least190%, at least 200%, at least 250%, at least 300%, at least 450%, or atleast 500%.

The disclosed compounds may exist as pharmaceutically acceptable salts.The term “pharmaceutically acceptable salt” refers to salts orzwitterions of the compounds which are water or oil-soluble ordispersible, suitable for treatment of disorders without undue toxicity,irritation, and allergic response, commensurate with a reasonablebenefit/risk ratio and effective for their intended use. The salts maybe prepared during the final isolation and purification of the compoundsor separately by reacting an amino group of the compounds with asuitable acid. For example, a compound may be dissolved in a suitablesolvent, such as but not limited to methanol and water and treated withat least one equivalent of an acid, like hydrochloric acid. Theresulting salt may precipitate out and be isolated by filtration anddried under reduced pressure. Alternatively, the solvent and excess acidmay be removed under reduced pressure to provide a salt. Representativesalts include acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,formate, isethionate, fumarate, lactate, maleate, methanesulfonate,naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate,propionate, succinate, tartrate, thrichloroacetate, trifluoroacetate,glutamate, para-toluenesulfonate, undecanoate, hydrochloric,hydrobromic, sulfuric, phosphoric and the like. The amino groups of thecompounds may also be quaternized with alkyl chlorides, bromides andiodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl,myristyl, stearyl and the like.

Basic addition salts may be prepared during the final isolation andpurification of the disclosed compounds by reaction of a carboxyl groupwith a suitable base such as the hydroxide, carbonate, or bicarbonate ofa metal cation such as lithium, sodium, potassium, calcium, magnesium,or aluminum, or an organic primary, secondary, or tertiary amine.Quaternary amine salts can be prepared, such as those derived frommethylamine, dimethylamine, trimethylamine, triethylamine, diethylamine,ethylamine, tributylamine, pyridine, N,N-dimethylaniline,N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine,dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine andN,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine,diethanolamine, piperidine, piperazine, and the like.

B. General Synthesis

The disclosed oxysterols may be prepared by synthetic processes or bymetabolic processes. Preparation of the compounds by metabolic processesincludes those occurring in the human or animal body (in vivo) orprocesses occurring in vitro.

The compounds and intermediates may be synthesiszed, isolated andpurified by methods well-known to those skilled in the art of organicsynthesis. Examples of conventional methods for isolating and purifyingcompounds can include, but are not limited to, chromatography on solidsupports such as silica gel, alumina, or silica derivatized withalkylsilane groups, by recrystallization at high or low temperature withan optional pretreatment with activated carbon, thin-layerchromatography, distillation at various pressures, sublimation undervacuum, and trituration, as described for instance in “Vogel's Textbookof Practical Organic Chemistry”, 5th edition (1989), by Furniss,Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical,Essex CM20 2JE, England.

A disclosed compound may have at least one basic atom or functionalgroup whereby the compound can be treated with an acid to form a desiredsalt. For example, a compound may be reacted with an acid at or aboveroom temperature to provide the desired salt, which is deposited, andcollected by filtration after cooling. Examples of acids suitable forthe reaction include, but are not limited to tartaric acid, lactic acid,succinic acid, as well as mandelic, atrolactic, methanesulfonic,ethanesulfonic, toluenesulfonic, naphthalenesulfonic, benzenesulfonic,carbonic, fumaric, maleic, gluconic, acetic, propionic, salicylic,hydrochloric, hydrobromic, phosphoric, sulfuric, citric, hydroxybutyric,camphorsulfonic, malic, phenylacetic, aspartic, or glutamic acid, andthe like.

Optimum reaction conditions and reaction times for each individual stepcan vary depending on the particular reactants employed and substituentspresent in the reactants used. Specific procedures are provided in theExamples section. Reactions can be worked up in the conventional manner,e.g. by eliminating the solvent from the residue and further purifiedaccording to methodologies generally known in the art such as, but notlimited to, crystallization, distillation, extraction, trituration andchromatography. Unless otherwise described, the starting materials andreagents are either commercially available or can be prepared by oneskilled in the art from commercially available materials using methodsdescribed in the chemical literature. Starting materials, if notcommercially available, can be prepared by procedures selected fromstandard organic chemical techniques, techniques that are analogous tothe synthesis of known, structurally similar compounds, or techniquesthat are analogous to the above described schemes or the proceduresdescribed in the synthetic examples section.

Routine experimentations, including appropriate manipulation of thereaction conditions, reagents and sequence of the synthetic route,protection of any chemical functionality that cannot be compatible withthe reaction conditions, and deprotection at a suitable point in thereaction sequence of the method are included in the scope of theinvention. Suitable protecting groups and the methods for protecting anddeprotecting different substituents using such suitable protectinggroups are well known to those skilled in the art; examples of which canbe found in PGM Wuts and T W Greene, in Greene's book titled ProtectiveGroups in Organic Synthesis (4^(th) ed.), John Wiley & Sons, NY (2006),which is incorporated herein by reference in its entirety. Synthesis ofthe compounds of the invention can be accomplished by methods analogousto those described in the synthetic schemes described hereinabove and inspecific examples.

When an optically active form of a disclosed compound is required, itcan be obtained by carrying out one of the procedures described hereinusing an optically active starting material (prepared, for example, byasymmetric induction of a suitable reaction step), or by resolution of amixture of the stereoisomers of the compound or intermediates using astandard procedure (such as chromatographic separation,recrystallization or enzymatic resolution).

Similarly, when a pure geometric isomer of a compound is required, itcan be obtained by carrying out one of the above procedures using a puregeometric isomer as a starting material, or by resolution of a mixtureof the geometric isomers of the compound or intermediates using astandard procedure such as chromatographic separation.

3. Pharmaceutical Compositions

The disclosed compounds may be incorporated into pharmaceuticalcompositions suitable for administration to a subject (such as apatient, which may be a human or non-human).

The pharmaceutical compositions may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of the agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may be determined by a person skilled in the art and mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the composition to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of a compound ofthe disclosure [e.g., an oxysterol] are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

For example, a therapeutically effective amount of a compound of adisclosed oxysterol may be about 1 mg/kg to about 1000 mg/kg, about 5mg/kg to about 950 mg/kg, about 10 mg/kg to about 900 mg/kg, about 15mg/kg to about 850 mg/kg, about 20 mg/kg to about 800 mg/kg, about 25mg/kg to about 750 mg/kg, about 30 mg/kg to about 700 mg/kg, about 35mg/kg to about 650 mg/kg, about 40 mg/kg to about 600 mg/kg, about 45mg/kg to about 550 mg/kg, about 50 mg/kg to about 500 mg/kg, about 55mg/kg to about 450 mg/kg, about 60 mg/kg to about 400 mg/kg, about 65mg/kg to about 350 mg/kg, about 70 mg/kg to about 300 mg/kg, about 75mg/kg to about 250 mg/kg, about 80 mg/kg to about 200 mg/kg, about 85mg/kg to about 150 mg/kg, and about 90 mg/kg to about 100 mg/kg.

The pharmaceutical compositions may include pharmaceutically acceptablecarriers. The term “pharmaceutically acceptable carrier,” as usedherein, means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as, but not limited to, lactose,glucose and sucrose; starches such as, but not limited to, corn starchand potato starch; cellulose and its derivatives such as, but notlimited to, sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as, but not limited to, cocoa butter and suppository waxes; oilssuch as, but not limited to, peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil and soybean oil; glycols; such aspropylene glycol; esters such as, but not limited to, ethyl oleate andethyl laurate; agar; buffering agents such as, but not limited to,magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol, and phosphatebuffer solutions, as well as other non-toxic compatible lubricants suchas, but not limited to, sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by, for example, soliddosing, eyedrop, in a topical oil-based formulation, injection,inhalation (either through the mouth or the nose), implants, or oral,buccal, parenteral, or rectal administration. Techniques andformulations may generally be found in “Remington's PharmaceuticalSciences”, (Meade Publishing Co., Easton, Pa.). Therapeutic compositionsmust typically be sterile and stable under the conditions of manufactureand storage.

The route by which the disclosed compounds are administered and the formof the composition will dictate the type of carrier to be used. Thecomposition may be in a variety of forms, suitable, for example, forsystemic administration (e.g., oral, rectal, nasal, sublingual, buccal,implants, or parenteral) or topical administration (e.g., dermal,pulmonary, nasal, aural, ocular, liposome delivery systems, oriontophoresis).

Carriers for systemic administration typically include at least one ofdiluents, lubricants, binders, disintegrants, colorants, flavors,sweeteners, antioxidants, preservatives, glidants, solvents, suspendingagents, wetting agents, surfactants, combinations thereof, and others.All carriers are optional in the compositions.

Suitable diluents include sugars such as glucose, lactose, dextrose, andsucrose; diols such as propylene glycol; calcium carbonate; sodiumcarbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. Theamount of diluent(s) in a systemic or topical composition is typicallyabout 50 to about 90%.

Suitable lubricants include silica, talc, stearic acid and its magnesiumsalts and calcium salts, calcium sulfate; and liquid lubricants such aspolyethylene glycol and vegetable oils such as peanut oil, cottonseedoil, sesame oil, olive oil, corn oil and oil of theobroma. The amount oflubricant(s) in a systemic or topical composition is typically about 5to about 10%.

Suitable binders include polyvinyl pyrrolidone; magnesium aluminumsilicate; starches such as corn starch and potato starch; gelatin;tragacanth; and cellulose and its derivatives, such as sodiumcarboxymethylcellulose, ethyl cellulose, methylcellulose,microcrystalline cellulose, and sodium carboxymethylcellulose. Theamount of binder(s) in a systemic composition is typically about 5 toabout 50%.

Suitable disintegrants include agar, alginic acid and the sodium saltthereof, effervescent mixtures, croscarmelose, crospovidone, sodiumcarboxymethyl starch, sodium starch glycolate, clays, and ion exchangeresins. The amount of disintegrant(s) in a systemic or topicalcomposition is typically about 0.1 to about 10%.

Suitable colorants include a colorant such as an FD&C dye. When used,the amount of colorant in a systemic or topical composition is typicallyabout 0.005 to about 0.1%.

Suitable flavors include menthol, peppermint, and fruit flavors. Theamount of flavor(s), when used, in a systemic or topical composition istypically about 0.1 to about 1.0%.

Suitable sweeteners include aspartame and saccharin. The amount ofsweetener(s) in a systemic or topical composition is typically about0.001 to about 1%.

Suitable antioxidants include butylated hydroxyanisole (“BHA”),butylated hydroxytoluene (“BHT”), and vitamin E. The amount ofantioxidant(s) in a systemic or topical composition is typically about0.1 to about 5%.

Suitable preservatives include benzalkonium chloride, methyl paraben andsodium benzoate. The amount of preservative(s) in a systemic or topicalcomposition is typically about 0.01 to about 5%.

Suitable glidants include silicon dioxide. The amount of glidant(s) in asystemic or topical composition is typically about 1 to about 5%.

Suitable solvents include water, isotonic saline, ethyl oleate,glycerine, hydroxylated castor oils, alcohols such as ethanol, andphosphate buffer solutions. The amount of solvent(s) in a systemic ortopical composition is typically from about 0 to about 100%.

Suitable suspending agents include AVICEL RC-591 (from FMC Corporationof Philadelphia, Pa.) and sodium alginate. The amount of suspendingagent(s) in a systemic or topical composition is typically about 1 toabout 8%.

Suitable surfactants include lecithin, Polysorbate 80, and sodium laurylsulfate, and the TWEENS from Atlas Powder Company of Wilmington, Del.Suitable surfactants include those disclosed in the C.T.F.A. CosmeticIngredient Handbook, 1992, pp.587-592; Remington's PharmaceuticalSciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1,Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. Theamount of surfactant(s) in the systemic or topical composition istypically about 0.1% to about 5%.

Although the amounts of components in the systemic compositions may varydepending on the type of systemic composition prepared, in general,systemic compositions include 0.01% to 50% of active [e.g., anoxysterol] and 50% to 99.99% of one or more carriers. Compositions forparenteral administration typically include 0.1% to 10% of actives and90% to 99.9% of a carrier including a diluent and a solvent.

Compositions for oral administration can have various dosage forms. Forexample, solid forms include tablets, capsules, granules, and bulkpowders. These oral dosage forms include a safe and effective amount,usually at least about 5%, and more particularly from about 25% to about50% of actives. The oral dosage compositions include about 50% to about95% of carriers, and more particularly, from about 50% to about 75%.

Tablets can be compressed, tablet triturates, enteric-coated,sugar-coated, film-coated, or multiple-compressed. Tablets typicallyinclude an active component, and a carrier comprising ingredientsselected from diluents, lubricants, binders, disintegrants, colorants,flavors, sweeteners, glidants, and combinations thereof. Specificdiluents include calcium carbonate, sodium carbonate, mannitol, lactoseand cellulose. Specific binders include starch, gelatin, and sucrose.Specific disintegrants include alginic acid and croscarmelose. Specificlubricants include magnesium stearate, stearic acid, and talc. Specificcolorants are the FD&C dyes, which can be added for appearance. Chewabletablets preferably contain sweeteners such as aspartame and saccharin,or flavors such as menthol, peppermint, fruit flavors, or a combinationthereof.

Capsules (including implants, time release and sustained releaseformulations) typically include an active compound (e.g., an oxysterol),and a carrier including one or more diluents disclosed above in acapsule comprising gelatin. Granules typically comprise a disclosedcompound, and preferably glidants such as silicon dioxide to improveflow characteristics. Implants can be of the biodegradable or thenon-biodegradable type.

The selection of ingredients in the carrier for oral compositionsdepends on secondary considerations like taste, cost, and shelfstability, which are not critical for the purposes of this invention.

Solid compositions may be coated by conventional methods, typically withpH or time-dependent coatings, such that a disclosed compound isreleased in the gastrointestinal tract in the vicinity of the desiredapplication, or at various points and times to extend the desiredaction. The coatings typically include one or more components selectedfrom the group consisting of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethylcellulose, EUDRAGIT coatings (available from Rohm & Haas G.M.B.H. ofDarmstadt, Germany), waxes and shellac.

Compositions for oral administration can have liquid forms. For example,suitable liquid forms include aqueous solutions, emulsions, suspensions,solutions reconstituted from non-effervescent granules, suspensionsreconstituted from non-effervescent granules, effervescent preparationsreconstituted from effervescent granules, elixirs, tinctures, syrups,and the like. Liquid compositions, which may be administered orally, mayinclude a disclosed oxysterol compound and a carrier, namely, a carrierselected from diluents, colorants, flavors, sweeteners, preservatives,solvents, suspending agents, and surfactants. Peroral liquidcompositions preferably include one or more ingredients selected fromcolorants, flavors, and sweeteners.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically include one or more of soluble filler substancessuch as diluents including sucrose, sorbitol and mannitol; and binderssuch as acacia, microcrystalline cellulose, carboxymethyl cellulose, andhydroxypropyl methylcellulose. Such compositions may further includelubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

The disclosed compounds can be topically administered. Topicalcompositions that can be applied locally to the skin may be in any formincluding solids, solutions, oils, creams, ointments, gels, lotions,shampoos, leave-on and rinse-out hair conditioners, milks, cleansers,moisturizers, sprays, skin patches, and the like. Topical compositionsinclude: a disclosed compound (e.g., an oxysterol), and a carrier. Thecarrier of the topical composition preferably aids penetration of thecompounds into the skin. The carrier may further include one or moreoptional components.

The amount of the carrier employed in conjunction with a disclosedcompound is sufficient to provide a practical quantity of compositionfor administration per unit dose of the medicament. Techniques andcompositions for making dosage forms useful in the methods of thisinvention are described in the following references: ModernPharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979);Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); andAnsel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

A carrier may include a single ingredient or a combination of two ormore ingredients. In the topical compositions, the carrier includes atopical carrier. Suitable topical carriers include one or moreingredients selected from phosphate buffered saline, isotonic water,deionized water, monofunctional alcohols, symmetrical alcohols, aloevera gel, allantoin, glycerin, vitamin A and E oils, mineral oil,propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castoroil, combinations thereof, and the like. More particularly, carriers forskin applications include propylene glycol, dimethyl isosorbide, andwater, and even more particularly, phosphate buffered saline, isotonicwater, deionized water, monofunctional alcohols, and symmetricalalcohols.

The carrier of a topical composition may further include one or moreingredients selected from emollients, propellants, solvents, humectants,thickeners, powders, fragrances, pigments, and preservatives, all ofwhich are optional.

Suitable emollients include stearyl alcohol, glyceryl monoricinoleate,glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil,cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate,isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate,decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate,di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropylstearate, butyl stearate, polyethylene glycol, triethylene glycol,lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylatedlanolin alcohols, petroleum, mineral oil, butyl myristate, isostearicacid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyllactate, decyl oleate, myristyl myristate, and combinations thereofSpecific emollients for skin include stearyl alcohol andpolydimethylsiloxane. The amount of emollient(s) in a skin-based topicalcomposition is typically about 5% to about 95%.

Suitable propellants include propane, butane, isobutane, dimethyl ether,carbon dioxide, nitrous oxide, and combinations thereof The amount ofpropellant(s) in a topical composition is typically about 0% to about95%.

Suitable solvents include water, ethyl alcohol, methylene chloride,isopropanol, castor oil, ethylene glycol monoethyl ether, diethyleneglycol monobutyl ether, diethylene glycol monoethyl ether,dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinationsthereof Specific solvents include ethyl alcohol and homotopic alcohols.The amount of solvent(s) in a topical composition is typically about 0%to about 95%.

Suitable humectants include glycerin, sorbitol, sodium2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate,gelatin, and combinations thereof Specific humectants include glycerin.The amount of humectant(s) in a topical composition is typically 0% to95%.

The amount of thickener(s) in a topical composition is typically about0% to about 95%.

Suitable powders include beta-cyclodextrins, hydroxypropylcyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums,colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammoniumsmectites, trialkyl aryl ammonium smectites, chemically-modifiedmagnesium aluminum silicate, organically-modified Montmorillonite clay,hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodiumcarboxymethyl cellulose, ethylene glycol monostearate, and combinationsthereof. The amount of powder(s) in a topical composition is typically0% to 95%.

The amount of fragrance in a topical composition is typically about 0%to about 0.5%, particularly, about 0.001% to about 0.1%.

Suitable pH adjusting additives include HCl or NaOH in amountssufficient to adjust the pH of a topical pharmaceutical composition.

In an embodiment, the pharmaceutical composition may include humanbreast milk. The active pharmaceutical ingredient may be a component ofhuman breast milk. The human breast milk may thus be administered to asubject in need of the active pharmaceutical ingredient.

In another embodiment, some infants may not be able to take food ormedication by mouth. For example, infants with acute brain injury arecritically ill and may not be able to take food or medication by mouth.Accordingly, total parenteral nutrition (TPN) may be administeredthrough a central line. TPN contains the hydration and nutrients neededto sustain life and grow the infant. Calories are delivered viacarbohydrates, protein, and lipids. The lipids may be administered as anintralipid emulsion. Oxysterol therapy may be added to the intralipidemulsion for intravenous administration, for example, through a centralline. An example of a commercially available intralipid emulsion is a20% fat emulsion containing soybean oil, egg yolk, phospholipids, andglycerin. Intralipid emulsions are commercially available.

4. Methods of Treatment

The disclosed oxysterols and compositions may be used in methods fortreatment of disorders and diseases related to brain injury, inparticular, injury to myelin. The methods of treatment may compriseadministering to a subject in need of such treatment a compositioncomprising a therapeutically effective amount of an oxysterol. Thesemethods promote the formation of oligodendrocytes, cells which functionto provide support and insulation to axons in the central nervous systemby creating the myelin sheath. Thus, the formation of oligodendrocytesmay serve to create myelin and repair damaged myelin in subjects withinjured myelin.

The compositions may be useful for treating and preventing certaindiseases and disorders in humans and animals related to myelin injury.Treatment or prevention of such diseases and disorders can be effectedby promoting oligodendrogenesis in a subject, by administering acompound or composition of the disclosure, either alone or incombination with another active agent as part of a therapeutic regimento a subject in need thereof.

Diseases and/or disorders which may be treated and/or prevented by thedisclosed methods include neonatal brain injury, traumatic brain injury,spinal cord injury, cerebral palsy, seizures, cognitive delay, multiplesclerosis, stroke, autism, leukodystrophy, schizophrenia and bipolardisorder. The neonatal brain injury may include at least one of diffusewhite matter injury, periventricular leukomalacia (PVL),hypoxic-ischemic encephalopathy (HIE), neonatal stroke, and grade 3-4intraventricular hemorrhages (IVH).

a. Neonatal Brain Injury

In humans, myelin development begins prenatally and continues into youngadulthood to achieve complex neurological functions. Failure of propermyelin development is a common pathology in neonatal brain injuryassociated with preterm and term births associated with hypoxic-ischemiaor other insults. The pathology, collectively called neonatal whitematter injury (WMI), ranges from diffuse non-necrotic changes affectingmainly subcortical white mater (hypoxic-ischemic encephalitis, HIE) tofocal lesions affecting periventricular white matter (periventricularleukomalacia, PVL), and is characterized by underdeveloped myelin.Afflicted children frequently present with diminished cognitive, motor,and psychiatric functions that persist beyond infancy.

b. Traumatic Brain Injury

Nearly two million people suffer traumatic brain injury in the US eachyear. These injuries adversely alter the metabolism of myelin.Furthermore, the loss of central myelinated nerve fibers continues overthe chronic post-traumatic phase after injury (Maxwell, W. Brain Sci.2013, 3(3), 1374-1394.).

c. Spinal Cord Injury

There are an estimated 10,000 to 12,000 spinal cord injuries every yearin the U.S. These injuries sever or crush the nerve fibers that runthrough the spinal cord, potentially leading to complete paralysis andloss of sensation below the level of the injury. A traumatic blow to thespinal cord also typically causes a loss of myelin and the death ofoligodendrocytes. Myelin is needed to insulate the electrical signalstransmitted by nerve fibers. Further, transplants of oligodendrocyteprecursor cells (OPCs) improve recovery in rats with spinal cord,underscoring the importance of remyelination in behavioral improvementafter a spinal cord injury.

d. Cerebral Palsy

Cerebral palsy is characterized by damage to myelin sheaths. When axonsbecome unmyelinated, they affect the ability of the brain to transmitsignals as efficiently as normal myelinated nerve cells. In turn, poorlytransmitted signals translate to an impaired nervous system. However,there are no efficient mechanisms within the human body for repairingmyelin sheaths around unmyelinated axons.

e. Seizures

Epilepsy is a chronic neurological disorder characterized by spontaneousrecurrent seizures, which also occur in demyelinating diseases of thecentral nervous system (CNS) with a higher prevalence. Demyelination hasbeen observed in the CNS of epilepsy patients, indicating an associationbetween demyelination and epileptic. A rat model that provides directevidence that myelin sheath damage in the rat brain started in the earlystage of epileptic seizures induction. Furthermore, myelination of axonsin children with epilepsy may be slowed by the epileptogenic process or,perhaps, there is seizure-related damage to the posterior corpuscallosum myelin sheath surrounding the onset of epilepsy.

f. Multiple Sclerosis

Multiple sclerosis (MS) is characterized by damage to the myelin sheath.The damage is caused by inflammation and the destruction of myelininterferes with nerve conduction. The symptoms of MS relate to thisinterruption of signaling between neurons. MS is the most common chronicdisabling disorder of the central nervous system in young adults. It maybe an autoimmune demyelinating disease in which an individual's immunesystem attacks their own body, in this case the myelin sheath in thebrain and spinal cord.

g. Stroke

The myelin sheath can be destroyed by stroke. If the sheath is able torepair and regenerate itself, normal nerve function may return. However,if the sheath is severely damaged, the underlying nerve fiber can die.Because nerve fibers in the CNS rarely regenerate, such damage remainsirreversible.

h. Autism

Abnormalities in white matter architecture have been described in humanautism. Region-specific decreases in myelin development observed inautism have been suggested to impact brain connectivity. Improvingmyelination may improve connectivity and mitigate autism-associatedsymptoms.

i. Leukodystrophy

Leukodystrophy is one of a group of disorders characterized bydegeneration of the white matter in the brain. The leukodystrophies arecaused by imperfect growth or development of the myelin sheath. Whendamage occurs to white matter, immune responses can lead to inflammationin the CNS, along with loss of myelin. Leukodystrophy is characterizedby specific symptoms including decreased motor function, musclerigidity, and eventually degeneration of sight and hearing. While thedisease is fatal, the age of onset is a key factor as infants are givena lifespan of 2 years, while adults typically live more than a decadeafter onset.

j. Schizophrenia

There is evidence for abnormalities in both myelin and axons amongpatients with schizophrenia, when compared with healthy individuals whounderwent the same testing. More specifically, there is a reduction inmyelination of white matter pathways in schizophrenia. Accordingly,myelination abnormalities in schizophrenia are associated withdisturbances in the functional integrity of the white matter.

k. Bipolar Disorder

Oligodendrocyte dysfunction and a loss of white matter have beenimplicated in bipolar disorder. Imaging studies have shown that patientswith bipolar disorder have less white matter than healthy patients.Post-mortem studies have demonstrated a cessation of development of newmyelin in early adulthood in patients with bipolar disorder, compared toa steady decrease over a lifetime in healthy patients.

Therefore, it would be beneficial to administer oxysterol therapy tosubjects who suffer from neonatal brain injury, traumatic brain injury,spinal cord injury, cerebral palsy, seizures, cognitive delay, multiplesclerosis, stroke, autism spectrum disorders, leukodystrophy,schizophrenia and bipolar disorder.

1. Modes of Administration

Methods of treatment may include any number of modes of administering adisclosed composition. Modes of administration may include tablets,pills, dragees, hard and soft gel capsules, granules, pellets, aqueous,lipid, oily or other solutions, emulsions such as oil-in-wateremulsions, liposomes, aqueous or oily suspensions, syrups, elixirs,solid emulsions, solid dispersions or dispersible powders. For thepreparation of pharmaceutical compositions for oral administration, theagent may be admixed with commonly known and used adjuvants andexcipients such as for example, gum arabic, talcum, starch, sugars (suchas, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-activeagents, magnesium stearate, aqueous or non-aqueous solvents, paraffinderivatives, cross-linking agents, dispersants, emulsifiers, lubricants,conserving agents, flavoring agents (e.g., ethereal oils), solubilityenhancers (e.g., benzyl benzoate or benzyl alcohol) or bioavailabilityenhancers (e.g. Gelucire®). In the pharmaceutical composition, the agentmay also be dispersed in a microparticle, e.g. a nanoparticulatecomposition.

For parenteral administration, the agent can be dissolved or suspendedin a physiologically acceptable diluent, such as, e.g., water, buffer,oils with or without solubilizers, surface-active agents, dispersants oremulsifiers. As oils for example and without limitation, olive oil,peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil maybe used. More generally spoken, for parenteral administration, the agentcan be in the form of an aqueous, lipid, oily or other kind of solutionor suspension or even administered in the form of liposomes ornano-suspensions.

In an embodiment, one or more oxysterol may be administered in acomposition comprising human breast milk. The human breast milk may thusbe administered orally to a subject in need of oxysterol therapy. Thehuman breast milk may be further supplemented with oxysterols inaddition to oxysterols that are naturally present in human breast milk.The oxysterols used for supplementation may be higher doses of one ormore oxysterols already present or they may be one or more oxysterolsnot found to be naturally occurring in human breast milk.

In another embodiment, one or more oxysterol may be administered in acomposition comprising infant formula. Infant formula is a manufacturedfood which purports to be or is represented for special dietary usesolely as a food for infants by reason of its simulation of human milkor its suitability as a complete or partial substitute for human milk.The infant formula may thus be administered orally to a subject in needof oxysterol therapy. The infant formula may further comprise at leastone oxysterol.

In addition, human breast milk or infant formula comprising at least oneoxysterol may be administered to prematurely born infants, regardless ofsuspected or known brain injury. Because oxysterols can be used topromote oligodendrogenesis and healthy myelin, breast milk or infantformula supplemented with additional amounts of oxysterols may bebeneficial to promoting brain development in prematurely born infants.

Infant formula which may be suitable for the methods described hereininclude, but are not limited to, milk-based formula (for example,SIMILAC®, ENFAMIL®, or GERBER GOOD START®), soy-based formula orlactose-free (for example, SIMILAC SOY ISOMIL®, ENFAMIL PROSOBEE®,GERBER GOOD START SOY®), partially or extensively hydrolyzed formulas(for example, ENFAMIL GENTLEASE®, NUTRAMIGEN®), and formula speciallydesigned for prematurely born infants (for example NEOSURE®, ENFACARE®).

m. Combination Therapies

Additional therapeutic agent(s) may be administered simultaneously orsequentially with the disclosed compounds and compositions. Sequentialadministration includes administration before or after the disclosedcompounds and compositions. In some embodiments, the additionaltherapeutic agent or agents may be administered in the same compositionas the disclosed compounds. In other embodiments, there may be aninterval of time between administration of the additional therapeuticagent and the disclosed compounds. In some embodiments, administrationof an additional therapeutic agent with a disclosed compound may allowlower doses of the other therapeutic agents and/or administration atless frequent intervals. When used in combination with one or more otheractive ingredients, the compounds of the present invention and the otheractive ingredients may be used in lower doses than when each is usedsingly. Accordingly, the pharmaceutical compositions of the presentinvention include those that contain one or more other activeingredients, in addition to an oxysterol. The above combinations includecombinations of a compound of the present invention not only with oneother active compound, but also with two or more other active compounds.

In certain embodiments, the oxysterol can be combined with a variety ofneuroprotective strategies. For example, in neonatal brain injury theonly approved therapy is whole body hypothermia. This therapy istypically administered within the first 6 hours of birth and iscontinued for 3 days. The infant is cooled to, for example, 33.5° C. for72 hours. After 72 hours, the infant is slowly rewarmed to 37° C., forexample. Oxysterol therapy could be combined with this whole bodyhypothermia to mitigate myelin injury and improve neurodevelopmentaloutcomes.

For the treatment of multiple sclerosis, oxysterols can be combined withstandard immunomodulatory therapies including, but not limited to,corticosteroids, beta interferon, glatiramer acetate, dimethyl fumerate,natalizumab, and mitoxantrone.

For the treatment of stroke, oxysterols can be combined with standardapproaches to lytic therapy including tissue plasminogen activator(tPA).

For the treatment of schizophrenia, oxysterols can be combined withantipsychotic therapy.

The disclosed compounds may be included in kits comprising the compound(e.g., one or more oxysterols), a systemic or topical compositiondescribed above, or both; and information, instructions, or both thatuse of the kit will provide treatment for medical conditions in mammals(particularly humans). The information and instructions may be in theform of words, pictures, or both, and the like. In addition or in thealternative, the kit may include the medicament, a composition, or both;and information, instructions, or both, regarding methods of applicationof medicament, or of composition, preferably with the benefit oftreating or preventing medical conditions in mammals (e.g., humans).

The compounds and processes of the invention will be better understoodby reference to the following examples, which are intended as anillustration of and not a limitation upon the scope of the invention.

n. Evaluation of Treatment

Quantification of oligodendrocyte cell numbers in the brain is criticalto determining the impact of oxysterol therapy. Stereology is a usefulresearch tool used by neuroscientists to provide accurate and unbiasedestimates of cell numbers within specified brain regions. The number ofoligodendrocyte numbers is determined using Stereo Investoigator™software (MBF Bioscience) and a Zeiss Axiolmager M2 motorizedfluorescent microscope with Apotome structured illumination. Perfusionfixed cryoprotected brains are frozen sectioned at 30mm and collectedfree-floating in phosphate buffered saline. Every 4^(th) caronal sectionbeginning at the appearance of the lateral ventricles and ending at thelevel of the anterior commissure are stained for olig2 and APC (CC1).Stained tissue is counter stained with DAPI and mounted on slides. UsingStereo Investigtor, the corpus callosum is outlined under lowmagnification. Olig2+APC+nucleated cells are then counted throughout thecorpus callosum using a 63× oil objective lens.

The detection of differences in locomotor function is an important toolfor the assessment of the severity of many conditions that affect thecentral nervous system (CNS), peripheral nervous system (PNS) andskeletal structures or muscles. A gait analysis system, such as theCatWalk™ XT, provides automatic and sensitive detection of a full rangeof parameters related to footprints and the dynamics of gait in animaltesting.

Traditionally, methods such as BBB scoring, running wheels, and ink onpaper are used. However, these are not always objective and quick tomaster, nor do they assess the temporal dynamics of gate accurately. TheCatWalk XT method is both accurate and objective, and allows for a moredetailed assessment of each individual footfall. CatWalk XT has provenitself as an objective, non-intrusive, and accurate tool for researchersin the field of CNS-related disease.

In particular, the regularity index, stand and step cycle, and swing andswing speeds may be measured utilizing these testing methods. Mice whichhave sustained myelin injury and are administered oxysterol therapy mayshow an improvement in regularity index, stand and step cycle, and swingand swing speeds over untreated mice. The improvement may be such foreach of these measurements that the values determined are similar or thesame as that of the values determined for control mice who havesustained no injured myelin.

6. EXAMPLES

20α-Hydroxycholesterol (20HC) and 22α-hydroxycholesterol were purchasedcommercially from Sigma-Aldrich. All oxysterols were resuspended in DMSOat 12mM for use in cell culture systems. In vivo studies utilizedoxysterols freshly dissolved in corn oil prior to administration.

Example 1 In vitro Oligodendrocyte Differentiation from Neural StemCells

To determine the impact of oxysterol exposure on cellulardifferentiation, primary neural stem cells from neonatal mice werecultured in chamber slides. Cells were cultured in media alone (control)or 1 μM 20α-hydroxycholesterol for 7 days were then allowed todifferentiate for 18 days. The differentiated cells were immunostainedfor markers of oligodendrocytes (Olig2 and CNPase) and visualized on aconfocal microscope (FIG. 1A). Larger magnification of FIG. 1A showsthat Olig2+CNPase+ cells have oligodendrocyte morphology (FIG. 1B). Cellcounting experiments demonstrated a significant increase inolig2+CNPase+ cells (oligodendrocytes) in the 20α-hydroxycholesteroltreated groups (p<0.02; FIG. 1C). Protein samples from control and20α-hydroxycholesterol treated stem cell cultures were then fractionatedand probed for markers of the oligodendrocyte lineage. Western blotanalysis revealed an increase in CNPase, Olig2 and myelin basic protein(MBP) in 20α-hydroxycholesterol treated cells (FIG. 1D).

Example 2 In vivo Oligodendrocyte Differentiation from Neural Stem Cells

20α-hydroxycholesterol was administered to uninjured postnatal miceduring the normal myelination period to determine if oxysterol exposureincreased the numbers of oligodendrocytes in vivo. Five day old micewere administered 4 daily doses of vehicle control (n=4) or20α-hydroxycholesterol (n=4) subcutaneously (100 mg/kg/day) and rested.Five days after the final injection, the mice were analyzed to quantifythe number of olig2+cells in the corpus callosum (FIG. 2A, 2B). Asignificant increase in olig2+cell population was observed in animalsreceiving 20α-hydroxycholesterol when compared to the control (p<0.02;FIG. 2C).

Taken together, these results show that 20α-hydroxycholesteroldemonstrated an ability to significantly promote oligodendrocytedifferentiation from neural stem cells both in vitro and in vivo.

Example 3 Perinatal Mouse Model of Myelin Injury

Perinatal bowel perforation is a common complication of premature birthand is strongly linked to myelin injury and cerebral palsy. Using aperinatal mouse model of bowel perforation to induce sepsis-associateddiffuse myelin injury on postnatal day 5, sepsis mice were determined tohave a 60% reduction of mature oligodendrocytes at postnatal day 25(FIG. 3).

Example 4 Efficacy of Oxysterol Therapy

To determine the efficacy of oxysterol therapy, the perinatal mousemodel was employed to induce injury in mice on postnatal day 5.Commencing 24 hours after injury, mice were administered daily singlesubcutaneous injections of 20α-hydroxycholesterol (100 mg/kg/day) for 5days (FIG. 3A). Coronal sections of the brains were analyzed usingstereology on postnatal day 25 to determine the number of olig2+APC+oligodendrocytes in the periventricular white matter of the corpuscallosum. Control, sepsis, and sepsis + oxysterol treated groups wereanalyzed by immunohistochemistry for olig2 and APC to identifyoligodendrocytes (FIG. 3B). Stereological counting revealed a reversalof oligodendrocyte injury in sepsis mice treated with oxysterol (FIG.3C, p=0.001). Accordingly, treatment with 20α-hydroxycholesterol insepsis-injured mice significantly increased the amount ofoligodendrocytes within the periventricular white matter. In fact,sepsis-injured mice treated with oxysterols had more oligodendrocytesthan vehicle treated control mice (FIG. 3B,C).

The impact of oxysterol therapy on motor function in injured mice wasalso determined. Mice were subjected to sepsis-induced myelin injury onpostnatal day 5. Twenty-four hours after injury, septic mice weredivided into a vehicle control group and an oxysterol treated group.Treatment with 20α-hydroxycholesterol (100 mg/kg/day) was carried outdaily for five days. At two months of age (adult), mice were evaluatedon a CatWalk Gait Analysis System (Noldus Inc.). The gait analysissystem identified multiple disturbances in the gait of untreated maturemice that survived perinatal sepsis. However, these gait disturbanceswere not present in mice treated with 20α-hydroxycholesterol.

Regularity Index: Regularity Index is a measure of interpawcoordination, and disturbances in regularity may be a result of cerebralinjury. The Regularity Index was determined for uninjured control mice(FIG. 4A,D), sepsis mice (FIG. 4B,D), and septic mice treated with theoxysterol 20HC (FIG. 4 C,D). FIG. 4 shows decreased regularity index foruntreated mature mice that survived perinatal sepsis when compared touninjured age-matched littermates. Remarkably, sepsis injuredlittermates treated with the oxysterol (20HC) completely reversed anyobserved deficits in Regularity Index (FIG. 4D).

Stand and step cycle: Stand and step cycle are measures of the length oftime the animal's paws are in contact with the platform as they walk.Increased stand and step cycle times are measures of gait instability.FIG. 5A and 5B demonstrate that sepsis injured mice possessed anincreased stand and step cycle, while those treated with the oxysterolresulted in similar values to the uninjured control mice.

Swing and swing speed: Swing and swing speed are measures of the animals“rocking” as they walk. While the swing was increased in perinatalsepsis survivors (FIG. 5C), the swing speed was decreased in sepsissurvivors (FIG. 5D) consistant with an unsteady gait. Administration of20α-hydroxycholesterol therapy reversed all of these measures of gaitdisturbances.

Example 5 Detection of Oxysterols in Human Breast Milk

Mass spectrometry assays were employed to identify oxysterols that arepresent in human breast milk. Samples of freshly pumped human breastmilk were obtained. Half of each sample was immediately frozen on dryice and stored at −80° C. The remainder was stored at 4° C. for six daysbefore analysis. Breast milk samples were then analyzed by massspectrometry and compared to oxysterols standards (FIG. 6A,B). While20α-hydroxycholesterol was not detected in human breast milk, abundantlevels of 24-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC),and 27-hydroxycholesterol (27HC) were observed (FIG. 7).

Example 6 In vitro Oligodendrocyte Differentiation from Neural StemCells with Breast Milk-Associated 24-hydroxycholesterol and25-hydroxycholesterol

Primary neural stem cells were treated with 24-hydroxycholesterol and25-hydroxycholesterol at 1 μm and 0.5 μm for five days, then allowed todifferentiate for 18 days. Protein lysates were probed foroligodendrocyte-associated proteins CNPase and myelin basic protein(MBP). Exposure of neural stem cells to these oxysterols inducedexpression of oligodendrocyte-associated proteins CNPase and MBP,suggesting similar activity as 20α-hydroxycholesterol (FIG. 8).

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the present disclosureare set out in the following numbered clauses:

Clause 1. A method of promoting oligodendrogenesis in a subject in needthereof, the method comprising administering a therapeutically effectiveamount of at least one oxysterol.

Clause 2. The method of clause 1, wherein the oxysterol promotesdifferentiation of neural stem cells into oligodendrocytes.

Clause 3. The method of clause 1 or clause 2, wherein the oxysterolcomprises a cholesterol derivative oxidized at any of carbons 20-27.

Clause 4. The method of any one of clauses 1-3, wherein the oxysterol isselected from the group consisting of: 20α-hydroxycholesterol;22(R)-hydroxycholesterol; 22(S)-hydroxycholesterol;24(R)-hydroxycholesterol; 24(S)-hydroxycholesterol;25-hydroxycholesterol; and 27-hydroxycholesterol; or a pharmaceuticallyacceptable salt thereof.

Clause 5. The method of clause 1, wherein the subject in need thereofsuffers from a disease or disorder associated with myelin injury.

Clause 6. The method of clause 5, wherein the disease or disorder isselected from at least one of neonatal brain injury, traumatic braininjury, spinal cord injury, cerebral palsy, seizures, autism spectrumdisorders, cognitive delay, multiple sclerosis, stroke, leukodystrophy,schizophrenia and bipolar disorder.

Clause 7. The method of clause 6, wherein the neonatal brain injury isselected from at least one of diffuse white matter injury,periventricular leukomalacia (PVL), hypoxic-ischemic encephalopathy(HIE), neonatal stroke, and grade 3-4 intraventricular hemorrhages(IVH).

Clause 8. The method of any one of clauses 1-7, wherein the oxysterolpromotes myelination.

Clause 9. A method of repairing injured myelin in a subject in needthereof, the method comprising administering a therapeutically effectamount of an oxysterol.

Clause 10. The method of clause 9, wherein the injured myelin is aresult of brain injury.

Clause 11. The method of clause 10, wherein the brain injury istraumatic brain injury or neonatal brain injury.

Clause 12. The method of clause 11, wherein the neonatal brain injury isselected from at least one of diffuse white matter injury,periventricular leukomalacia (PVL), hypoxic-ischemic encephalopathy(HIE), neonatal stroke, and grade 3-4 intraventricular hemorrhages(IVH).

Clause 13. The method of any one of clauses 9-12, wherein the oxysterolpromotes differentiation of neural stem cells into oligodendrocytes.

Clause 14. The method of any one of clauses 9-13, wherein the oxysterolcomprises a cholesterol derivative oxidized at any of carbons 20-27.

Clause 15. The method of any one of clauses 9-14, wherein the oxysterolis selected from the group consisting of: 20α-hydroxycholesterol;22(R)-hydroxycholesterol; 22(S)-hydroxycholesterol;24(R)-hydroxycholesterol; 24(S)-hydroxycholesterol;25-hydroxycholesterol; and 27-hydroxycholesterol; or a pharmaceuticallyacceptable salt thereof.

Clause 16. A pharmaceutical composition comprising at least oneoxysterol and at least one pharmaceutically acceptable carrier.

Clause 17. The pharmaceutical composition of clause 16, furthercomprising human breast milk.

1. A method of promoting oligodendrogenesis in a subject in needthereof, the method comprising administering a therapeutically effectiveamount of at least one oxysterol.
 2. The method of claim 1, wherein theoxysterol promotes differentiation of neural stem cells intooligodendrocytes.
 3. The method of claim 1, wherein the oxysterolcomprises a cholesterol derivative oxidized at any of carbons 20-27. 4.The method of claim 1, wherein the oxysterol is selected from the groupconsisting of: 20 α-hydroxychole sterol; 22(R)-hydroxycholesterol;22(S)-hydroxycholesterol; 24(R)-hydroxycholesterol;24(S)-hydroxycholesterol; 25 -hydroxychole sterol; and27-hydroxycholesterol; or a pharmaceutically acceptable salt thereof. 5.The method of claim 1, wherein the subject in need thereof suffers froma disease or disorder associated with myelin injury.
 6. The method ofclaim 5, wherein the disease or disorder is selected from at least oneof neonatal brain injury, traumatic brain injury, spinal cord injury,cerebral palsy, seizures, autism spectrum disorders, cognitive delay,multiple sclerosis, stroke, leukodystrophy, schizophrenia and bipolardisorder.
 7. The method of claim 6, wherein the neonatal brain injury isselected from at least one of diffuse white matter injury,periventricular leukomalacia (PVL), hypoxic-ischemic encephalopathy(HIE), neonatal stroke, and grade 3-4 intraventricular hemorrhages(IVH).
 8. The method of claim 1, wherein the oxysterol promotesmyelination.
 9. A method of repairing injured myelin in a subject inneed thereof, the method comprising administering a therapeuticallyeffect amount of an oxysterol.
 10. The method of claim 9, wherein theinjured myelin is a result of brain injury.
 11. The method of claim 10,wherein the brain injury is traumatic brain injury or neonatal braininjury.
 12. The method of claim 11, wherein the neonatal brain injury isselected from at least one of diffuse white matter injury,periventricular leukomalacia (PVL), hypoxic-ischemic encephalopathy(HIE), neonatal stroke, and grade 3-4 intraventricular hemorrhages(IVH).
 13. The method of claim 9, wherein the oxysterol promotesdifferentiation of neural stem cells into oligodendrocytes.
 14. Themethod of claim 9, wherein the oxysterol comprises a cholesterolderivative oxidized at any of carbons 20-27.
 15. The method of claim 9,wherein the oxysterol is selected from the group consisting of:20α-hydroxycholesterol; 22(R)-hydroxycholesterol;22(S)-hydroxycholesterol; 24(R)-hydroxycholesterol;24(S)-hydroxycholesterol; 25-hydroxycholesterol; and27-hydroxycholesterol; or a pharmaceutically acceptable salt thereof 16.A pharmaceutical composition comprising at least one oxysterol and atleast one pharmaceutically acceptable carrier.
 17. The pharmaceuticalcomposition of claim 16, further comprising human breast milk.